br Materials and methods br

Materials and methods
Data and statistical analysis The results were expressed as mean ± SEM. One-way analysis of variance (ANOVA), followed by Bonferroni\'s post-test or unpaired Student\'s t-test were used to determine significant differences between groups. The program used for statistical analysis was graph pad prism software version 6.0. Differences were considered significant when p ≤ 0.05.
Results
Discussion The antihyperglycemic effect of BA, as well as its mechanism of insulinomimetic action in skeletal muscle, has been partially described to involve GLUT4 translocation, phosphoinositide-3-kinase (PI3K) and mitogen-activated protein kinases (MAPKs) activation as extracellular regulated kinase (ERK) mitogen-activated protein kinase (MEK)/extracellular regulated kinase (ERK) [4]. Furthermore, BA has been described as an insulin-secreting agent in hyperglycemic animals treated in vivo [4,27]; however, its mechanism of action for insulin secretion has not been characterized yet. The influx of calcium in β-pancreatic Betaine is a determinant factor for insulin secretion. Thus, compounds that can stimulate this influx directly or indirectly are potential secretagogues of insulin and, thus, antidiabetics. The stimulation of the influx of calcium in pancreatic islets by BA within 15 min (Fig. 1) corroborates its reported effect on the significant potentiation of glucose-mediated insulin secretion in vivo after 30 min of the induction of hyperglycemia [4]. This increase in calcium influx indicates that BA can stimulate insulin secretion by glucose-like pathways. This hypothesis was confirmed using the pharmacological activator of KATP channels, diazoxide (Fig. 2A). The inhibition of BA stimulated-calcium influx with diazoxide shows that this triterpene may exhibit a modulatory action on KATP channels, as corroborated by the stimulating effect of glibenclamide on pancreatic islets (Fig. 2B). Sulphonylureas, like glibenclamide, bind to the channel\'s SUR1 subunit regulatory portion, inhibiting potassium efflux. Furthermore, KATP can also be inhibited by ATP binding to the Kir 6.2 subunit, the channel responsible for internal rectification, by simply changing the Mg2+-ADP (activator) complex to ATP [28]. Thus, BA could stimulate calcium influx by a direct interaction with KATP channels or through indirect mechanisms leading to the inhibition or stimulation of pathways that modulate this channel, although an interaction between BA and KATP has not been described yet (Fig. 2A and B). This KATP-dependent BA effect, coupled with the inhibition of the stimulatory action of this triterpene on calcium influx in pancreatic islets with nifedipine suggests that this triterpene acts by stimulating cellular depolarization followed by activation of the VDCCs (Fig. 2C), similarly to the glucose-mediated mechanism [29]. However, the partial inhibition of the effect of BA by nifedipine indicates a probable participation of other ionic channels or signaling pathways in the influx of calcium and/or insulin secretion, such as L-VDCC (Fig. 2C), ClCs or intracellular pathways mediated by kinases [6,7,11,30]. The involvement of the chloride channels in insulin secretion is still not well understood. However, the main insulin secretagogue, glucose, reportedly stimulates insulin secretion and electrical activity even when the KATP channel is inhibited by diazoxide [31]. This indicates the existence of mechanisms of insulin secretion, induced by glucose, that involve electrophysiological events independent of potassium channels. Interestingly, we found that the mechanism of action of BA is also dependent on the activity of ClCs and CaCCs (Fig. 3A and B). In a model of glucose-induced insulin secretion, the stimulation of ClCs and CaCCs can occur regardless of the action of KATP channels, by the activation of kinases such as PKC and PKCaMII, and/or due to the increase in calcium influx [5,6]. The results described above using the inhibitors, 9-AC (Fig. 3B) and nifedipine (Fig. 2C), corroborate the involvement of CaCCs in the stimulatory effect of BA since events that increase intracellular calcium content are strongly associated with the activation of CaCCs. These events can involve the activity of ClCs, the release of calcium from intracellular stores via inositol 1,4,5 trisphosphate (IP3) signaling, the binding to G protein-coupled receptors and/or CaMKII activation [[32], [33], [34]]. However, this is the first report of a mechanism of action of a triterpene on chloride channels in the literature [35,36].